Genome-wide association studies have highlighted the need for tools to quantify the expression of genes in an allele-specific manner to show how disease-associated single nucleotide variants (SNVs) affect transcription. Advances in single cell imaging have enabled researchers to detect individual RNAs with single molecule resolution (Femino et al., 1998, Science 280:585-590; Raj et al., 2008, Nat Methods 5:877-879), or in conjunction with single chromosomes (Levesque and Raj, 2013, Nat Methods, doi:10.1038/nmeth.2372). However, such methods are typically unable to distinguish SNVs in these molecules, and the few methods available for in situ SNV detection tend to be complex and suffer from low efficiency (Larsson et al., 2004, Nat Methods 1:227-232; Larsson et al., 2010, Nat Methods 7:395-397). In one method, a complex enzymatic scheme was used to amplify signals from SNVs on RNA molecules to the point where SNVs could be detected in situ (Larsson et al., 2010, Nat Methods 7:395-397). However, the detection efficiency of this method was very low, likely on the order of 1% or lower in many cases. Accordingly, development of a method for SNV detection that is able to measure allele-specific gene expression at the single-cell and single-molecule level would be of great utility in a variety of fields, including genetics and transcription (Ferguson-Smith, 2011, Nat Rev Genet 12:565-575; Gimelbrant et al., 2007, Science 318:1136-1140; Gregg et al., 2010, Science 329: 643-648).
One of the primary difficulties associated with detecting a difference of a single base via RNA fluorescence in situ hybridization (FISH) is that an oligonucleotide probe on the order of 20 bases or more often hybridizes to the RNA despite the presence of a single mismatch. On the other hand, very short oligonucleotide probes, while able to discriminate between single-base differences, often fail to remain bound to the target due to reduced binding energy. In either case, distinguishing legitimate signals from false positives can be problematic when using just a single probe.
Thus, there is a need in the art for a method for SNV detection that can efficiently measure gene expression at the population, single cell or single molecule level. The present invention addresses this unmet need in the art.